The broad long term objective of this proposal is to understand the pathogenesis of natural killer (NK) LGL leukemia. The mechanisms responsible for expansion of leukemic NK cells are not completely known. Leukemic NK cells are characterized by Fas-resistance. Data supported by this grant has indicated that constitutively active RAS/MEK/ERK MAPK signaling contributes to the survival of leukemic NK cells. The central hypothesis of this proposal is that sphingolipid signaling as an upstream activator of RAS/MEK/ERK MAPK mediates leukemic NK cell survival. Our overall strategy will utilize a genetic approach to inhibit key pathway components in the specific aim. We anticipate that targeting these signaling cascades will reverse Fas-resistance in vitro and have therapeutic efficacy in an in vivo model of NK-LGL leukemia. Preliminary data indicate that many of the core genes central to sphingolipid metabolism such as acid ceramidase 1 (AC1) and sphingosine kinase 1(SPHK1) are differentially overexpressed in leukemic NK cells. We will test the hypothesis that alterations of ceramide/S1P rheostat contribute to Fas-resistance (Specific Aim). Pharmacological inhibition of either AC or SPHK1 led to apoptosis in leukemic NK cells. We found that S1P and dihydro-S1P levels were elevated in NK-LGL patients'sera. Furthermore, S1P protected leukemic NK cells from Fas-mediated apoptosis. AC and SPHK1 will be further evaluated as potential therapeutic targets of sphingolipid metabolism. Compelling preliminary data indicate that these key survival pathways are also activated in normal NK cells by target binding in vitro. Mechanistic investigations as outlined in this proposal will identify novel therapeutic targets for NK-LGL leukemia and also provide insight into survival mechanisms utilized by an activated innate immune system.